Cloning,Expression,Purification And Properties Of Nitrite Reductase From Lactobacillus Plantarum DMDL9010

Nitrite is usually used as a food additive added to the pickled food and fermented one, taking too excessive nitrites will induce methemoglobin, and nitrite is a precursor of the nitrosamines, which is a kind of carcinogenic substance, it can induce many kinds of cancer in humen’s digestive system, such as gastric cancer, colorectal cancer and liver cancer, and so on. Therefore nitrite is a kind of potential carcinogen and it is very necessary to control nitrite amount in the food. Nitrite reductase(NIR) can degrade nitrites effectively. One strain lactic acid bacteria DMDL9010 with the strong activity of nitrite gradation was isolated from Chinese paocai and identified as Lactobacillus plantarum in our previous research. On this basis of Lactobacillus plantarum DMDL9010, the gene cloning of Ni R was carried out and prokaryotic recombinant plasmid that contains the gene of NIR was built. Then great quantities of recombinant protein NIR(r NIR) was expressed by means of inducing the recombinant strains. And the r NIR protein was purified by using affinity chromatography. Moreover the enzym properties of r NIR was investigated. The main results were as follows:Firstly, we found the DNA sequence of the NIR of the Lactobacillus plantarum DMDL9010 in the NCBI, the size of this sequence is 1638 bp. According to this sequence, we designed two primers, and the fragment of nir was amplified by polymerase chain reaction(PCR) with the DNA of Lactobacillus plantarum DMDL 9010 as template. After the fragment was sequenced, the size of the nir fragment was about 1700 bp. The nir fragment and the plasmid p ET-32a(+) were cut by double digests, then the nir fragment was connected to the plasmid p ET-32a(+) and transferred into E. coli DH5α, and the recombinant plasmid was tested by PCR and double enzyme orientation, finally the recombinant plasmid p ET-32a(+)-nir was constructed successfully.The recombinant plasmid p ET-32a(+)-nir was transferred into E. coli BL21(DE3), and the after the recombinant strain p ET-32a(+)-nir-BL21 can degrade 45 μg/m L Na NO2 in the medium. The recombinant strain p ET-32a(+)-nir-BL21 was inoculated into the LB medium which was included 50 μg/m L ampicillin, and fermented in 37 °C, 180 rpm. When the OD600 of the fermented liquid was about 0.5, the IPTG was added into the fermented liquid and it was fermented in 30 °C, 180 rpm for 4 h. The crude protein was collected by the ways of ultrasonic broken and centrifugal, and the recombinant protein was collected by the way of nickel affinity chromatography, the high purity of r NIR was got, the expression of the r NIR in the recombinant strain was 82.01 mg/L, and the size of the r NIR was about 60 k D which was test by SDS-PAGE.The rNIR can’t degrade the nitrite without electron donor. When the electron donor I or II was added, the r NIR can degrade the nitrite effectively. The most suitable electron donor of the r NIR was cytochrome C, and it’s optimal concentration was 0.05 mg/m L in the reaction system. The optimum reaction time of the r NIR was 24 h, the optimum reaction temperature was 37 °C and the optimal reaction substrate concentration was 50 mg/L, the optimal buffer was HEPES buffer(50 m M HEPES, 150 m M Na Cl, p H 7.4).